Proteins catalyzing the extension of plant cell walls

ABSTRACT

Plant cell expansion is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive &#34;wall loosening&#34; enzymes. By employing a reconstitution approach, we initially found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicots and monocots, but was less effective on grass coleoptile walls. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD, as measured by SDS-PAGE, associated with such activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall We proposed the name &#34;expansins&#34; for this class of proteins. Expansins have been isolated from various plant sources including oat, cucumber, broccoli, celery, tomato, cotton, cabbage, and corn, and also from snail and its feces. These proteins weaken the intermolecular bonds between plant wall polysaccharides. They decrease the mechanical strength of commercial products made from polysaccharides, such as paper, and therefore present a novel approach in developing new technologies in industries which make use of such polysaccharides, such as in the paper industry, in the applications of polysaccharide gums and related products. These proteins moreover present a novel approach in the control of plant growth.

This application is a continuation-in-part of application Ser. No.08/242,090 filed on May 12, 1994, now abandoned which is a continuationin-part application of U.S. Ser. No. 08/060,944 filed May 12, 1993 (nowabandoned).

BACKGROUND OF THE INVENTION

The present invention relates to a new class of proteins, known asexpansins, and isolation and utilization of same. These proteins havebeen identified in a wide variety of plant materials and have a varietyof applications, including but not limited to, agricultural, food andindustrial uses, such as use in paper industry as a catalyst forweakening the strength of paper products. For example, they can proveespecially useful in the recycling of paper.

By the way of background, for many years wall "loosening enzymes" havebeen implicated in the control of plant cell enlargement (growth),largely on the basis of rapid biophysical and biochemical changes in thewall during auxin-induced growth (reviewed by Cleland and Rayle, Bot.Mag. Tokyo, 1:125-139, 1978; Taiz, Annu. Rev. Plant Physiol.,35:585-657, 1984). Plant walls contain numerous hydrolytic enzymes,which have been viewed as catalysts capable of weakening the wall topermit turgor-driven expansion (reviewed by Fry, Physiol. Plantarum,75:532-536, 1989). In support of this hypothesis, Huber and Nevins(Physiol. Plant., 53:533-539, 1981) and Inoue and Nevins (PlantPhysiol., 96:426-431, 1991) found that antibodies raised against wallproteins could inhibit both auxin-induced growth and wall autolysis ofcorn coleoptiles. In addition, isolated walls from many species extendirreversibly when placed under tension in acid conditions in a mannerconsistent with an enzyme-mediated process (Cosgrove D. J. Planta,177:121-130, 1989). Despite these results and other evidence in favor of"wall-loosening" enzymes, a crucial prediction of this hypothesis hasnever been demonstrated, namely that exogenously added enzymes or enzymemixtures can induce extension of isolated walls. To the contrary,Ruesink (Planta, 89:95-107, 1969) reported that exogenous wallhydrolytic enzymes could mechanically weaken the wall withoutstimulating expansion. Similarly, autolysis of walls during fruitripening does not lead to cell expansion. Thus a major piece of evidencein favor of wall-loosening enzymes as agents of growth control has beenlacking.

The walls of growing cucumber seedlings possess extractable proteinswhich can induce extension of isolated walls. Additionally, we identifytwo specific wall-associated proteins with this activity. We propose thename "expansin" for this class of proteins, defined as endogenous cellwall proteins which restore extension activity to inactivated walls heldunder tension. We further propose the specific names "expansin-29" and"expansin-30" (abbreviated Ex-29 and Ex-30, with respect to theirrelative molecular masses; McQueen-Mason et al. Plant Cell, 4:1425-1433,1992) for the two proteins isolated from cucumber. More recently we haveidentified an oat coleoptile wall protein that induced extension inisolated dicot walls (Z. C. Lee et al., 1993, Planta, 191:349-356). Theoat protein has an apparent molecular mass of 29 kD as revealed bySDS-PAGE. For clarity we will refer to the cucumber proteins as cEx, andto the oat proteins oEx. New data demonstrate that an expansin-likeprotein may be found in proteins obtained from the digestive track ofsnail and its feces. We will refer to the snail protein as sEx.

During studies of the biochemical mechanism of action of expansins, wefound that they had the ability to weaken the hydrogen bonding betweenplant cell wall polysaccharides (such as cellulose fibers). Thesefindings allow us to propose the following commercial uses of thesenovel proteins, including paper treatment, agricultural uses, and avariety of use in food and industrial markets.

The paper products industry employs 3/4 million workers and is a$60-billion industry in the U.S. alone (plus $40 billion in retailsales). Recycling is a growing concern and will prove more important asthe nation's landfill sites become more scarce and more expensive.According to 1992 testimony before congress on the problems andopportunities of paper recycling, the need for improved technology inpaper recycling is urgent and of high priority. The use of expansins inthis industry may be well received at this time.

Paper derives its mechanical strength from hydrogen bonding betweenpaper fibers, which are composed primarily of cellulose. During paperrecycling, the hydrogen bonding between paper fibers is disrupted bychemical and mechanical means prior to re-forming new paper products.Expansins may be used to weaken the hydrogen bonding between the paperfibers of recycled paper. As demonstrated in this invention disclosure,expansins, at very low concentrations, in fact weaken commercial papers,including slick paper from magazines and catalogs. These latter types ofpapers are difficult to recycle because they are not easily disrupted incommercial recycling processes.

The advantages of using expansins for paper recycling include thefollowing: the protein is nontoxic and environmentally innocuous; itcould substitute for current harsh chemical treatments which areenvironmentally noxious. The protein is effective on paper productswhich are now recalcitrant to current recycling processes. Its use couldexpand the range of recyclable papers. Because the protein acts atmoderate temperature and in mild chemical environments, degradation ofpaper fibers during recycling should be reduced. This should allow forrecycled paper fibers with stronger mechanical properties and with theability to be recycled more often than is currently practical. Moreover,savings in energy costs associated with heating and beating the papermay be realized.

Other modes of application of expansins includes production of virginpaper. Pulp for virgin paper is made by disrupting the bonding betweenplant fibers. Following the reasoning listed above, expansins may beuseful in the production of paper pulp from plant tissues. Use ofexpansins could substitute for harsher chemicals now in use and therebyreduce the financial and environmental costs associated with disposingof these harsh chemicals. The use of expansins could also result inhigher quality plant fibers because they would be less degraded thanfibers currently obtained by harsher treatments.

SUMMARY OF THE INVENTION

In accordance with the present invention, a new class of proteins andmethods related thereto are presented. The proteins which can becharacterized as catalysts of the extension of plant cell walls and theweakening of the hydrogen bonds in the pure cellulose paper are referredto as expansins. In general, the present invention is a compositioncomprising an isolated, purified and salt-soluble polypeptide in an acidmedium, wherein the polypeptide has an amino acid sequence with amolecular weight of about 29-30 kD as measured by SDS-PAGE, and inducesexpansin of inert plant wall material. Preferably, the pH of the acidmedium is in the range of 3.5-5.5 and additionally may comprise asulfhydryl reducing agent. The pH range is more preferably about 4-5 andmost preferably is about 4.5.

Two proteins have been isolated by fractionation techniques from washedwall fragments of the cucumber hypocotyls, referred to as "cucumberexpansin-29" and "cucumber expansin-30" (abbreviated cEx-29 and cEx-30,with respect to their relative masses). Another expansin protein hasbeen isolated from oat coleoptiles (oat expansin oEx-29). Expansinsappear to be broadly distributed throughout the plant kingdom and havebeen identified in stem and leaf vegetables (i.e., broccoli, cabbage),fruit and seed vegetables (i.e., tomato), fiber crops and cereals (i.e.,corn), and forest and ornamental crops (i.e., cotton). Also,expansin-like protein has been found in proteins obtained from thedigestive track of snail and its feces (sEx). These novel proteins canfind use in a variety of applications including the paper industry inproduction of paper pulp in preparation of virgin paper and in paperrecycling as a preferred way of disruption of paper fibers due to theirnontoxicity and environmental innocuosity in contrast with the harshchemical treatments applied today, which are environmentally noxious.

OBJECTS OF THE INVENTION

An object of this invention is to provide novel extractable proteinswhich can induce extension of isolated walls and can weaken commercialpaper products.

Another object of this invention is to provide a method of isolation ofthe said proteins from various sources including growing plant tissue.

It is also an object of this invention to develop methods of using thesaid proteins. These and other objects and advantages of the inventionover the prior art and a better understanding of its use will becomereadily apparent from the following description and are particularlydelineated in the appended claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Extension curves of native and reconstituted isolated cell wallspecimens obtained using a constant load extensometer.

Ten-mm sections of tissue were frozen and thawed, and lightly abradedprior to suspension in the extensometer. Tissues were clamped under anapplied force of 20 g and extension recorded using a linear voltagedisplacement transducer. The length of tissue between the clamps was 5mm.

(A) Extension curves of native and reconstituted cucumber hypocotylwalls. From top to bottom: native walls were suspended under tension in50 mM Hepes, pH 6.8, for 20 min. after which the bathing solution wasreplaced by 50 mM sodium acetate, pH 4.5, (arrow). Inactivated wallswere treated for 15 sec with boiling water prior to suspension; as shownin the second line, this treatment eliminated acid induced extension.For reconstitution experiments, inactivated walls were suspended in 50mM sodium acetate, pH 4.5, for 30 min. at which point the bathingsolution was replaced by 0.5 mL of fresh solution (arrows) containing2-3 mg of proteins extracted from growing cell walls (apical wallprotein), or with soluble proteins from growing cells (apical solubleproteins), or proteins extracted from cell walls from the nongrowingcotyledon (cotyl. wall protein) or from walls of the basal hypocotyl(basal wall protein).

(B) Reconstitution of extension activity in inactivated (heat treated)walls from growing tissues o f different species by the active cucumberwall extract. Walls from the growing region from tomato, pea and radishhypocotyls, lily and onion leaves, and coleoptiles of maize and barleywere prepared, heat-treated, and suspended as given above, except that aforce of only 10 g was applied to the more fragile tomato and radishwalls. Walls were first suspended in 50 mM sodium acetate, pH 4.5, after30 min. (arrow) the bathing solution was replaced with 0.5 mL of thesame buffer containing 2-3 mg of active wall protein from cucumberhypocotyls (apical 3 cm).

FIG. 2. Fractionation of extension-inducing cucumber wall extracts byHPLC.

(A) Ammonium sulfate precipitates of salt extracted cucumber wallextracts were resuspended and loaded onto a C3 hydrophobic interactionscolumn in 50 mM sodium acetate, pH 4.5, 20% (saturation) ammoniumsulfate. Proteins were eluted in a descending gradient (20-0%) ofammonium sulfate. Fractions were desalted and checked for extensioninducing activity with inactivated cucumber tissues as in FIG. 1. A₂₈₀nm is shown by a solid line and extension inducing activity by a brokenline.

(B) Active fractions from (A) were concentrated and desalted into 15 mMMes, pH 6.5, and loaded onto a sulfopropyl anion exchange columnequilibrated with the same buffer. Proteins were eluted with anascending gradient of NaCl (0-1 M) in this buffer. Extension inducingactivity (broken line) of fractions was checked directly after adjustingthe pH to 4.5 with 1 M acetic acid.

FIG. 3. SDS-PAGE of ammonium sulfate precipitate (AS), and activefractions from C3 and sulfopropyl (S1 and S2) HPLC separations ofcucumber proteins.

Protein samples were concentrated, desalted, and run onSDS-polyacrylamide gels according to the method of Laemmli. Gels werestained with Coomassie Brilliant Blue R 250. Arrows indicate the majorprotein bands at 29 and 30 kD in the S1 and S2 fractions which appear topossess the extension-inducing activity.

FIG. 4. The effects of DTT, metal ions, methanol and water boiling, andprotease treatments on reconstituted extension.

(A) The effect of DTT on reconstituted extension. Growing wall specimensof cucumber hypocotyl were inactivated by heat and then reconstituted byshaking in a solution of active C3 proteins (estimated concentration of50 micrograms per mL). Walls were then clamped under constant load (asdescribed in FIG. 1) firstly in a bathing solution of 50 mM Hepes, pH6.8. After 20 min. the solution was changed for 50 mM sodium acetate, pH4.5 (first arrow). After a further 40 min. DTT (from a 100 mM stocksolution) was added to give a final concentration of 10 mM. The firsttwo lines represent typical traces from 4 experiments with and withoutDTT addition. For the effects of metal ions, growing wall specimens fromcucumber hypocotyl were inactivated by heat and then clamped underconstant load (as described in FIG. 1) in a bathing solution of 50 mMsodium acetate, pH 4.5. After 20 min. the bathing solution was exchangedfor a fresh one containing 50 micrograms per mL of active C3 proteins(first arrow). After a further 40 min. AlCl₃.6H₂ O or CuCl₂.2H₂ O (from100 mM stock solutions) was added to bring the bathing solution to afinal concentration of I mM (second arrow). All experiments wererepeated 4 times. Line 3 shows extension without the addition of metalions, the next two lines show typical data obtained with the addition ofAlCl₃ and CuCl₂ respectively.

For the effects of boiling cell walls in methanol or boiling in water onthe recovery of extension inducing activity, growing cucumber hypocotyltissue was first boiled for 3 min. in methanol or for 30 sec indistilled water, wall fragments were recovered, cleaned and extracted(as described in FIG. 1). Proteins were precipitated with (NH₄)₂ SO₄)and resuspended in 50 mM sodium acetate, pH 4.5 before being tested inthe reconstitution assay described in FIG. 1. Lines 6 and 7 arerepresentative data from 4 experiments. (B) Growing wall specimens wereinactivated by heat and reconstituted with C3 proteins. Reconstitutedwalls were incubated with 1000 units of trypsin or chymotrypsin for 4 hrat 30° C., in 50 mM Hepes, pH 7.3, or with 2 milligrams per mL ofpronase or papain for 4 hr at 30° C., in 50 mM sodium acetate, pH 5.0.Controls were reconstituted and incubated in the same manner without theaddition of proteases. At the end of the incubations tissues wereclamped under constant load (as described in FIG. 1), first in 50 mMHepes, pH 6.8. After 30 min. the bathing solution was replaced by 50 mMsodium acetate, pH 4.5. The difference in the two rates of extension wascalculated. Data presented are the means of four experiments in eachcase.

FIG. 5. Effect of pH on reconstituted extension.

Cucumber wall specimens, inactivated by heat, were clamped underconstant load (as described in FIG. 1). Initial bathing solutions were50 mM citric acid, titrated to pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or6.5 with 1 M K₂ HPO₄. After 30 min. the bathing solution was changed fora 1:1 dilution of active C3 proteins with the relevant buffer (finalestimated protein concentration was 50 micrograms per mL), wherenecessary the final pH was adjusted using either 1 M citric acid or 1 MK₂ HPO₄. Extension was recorded for a further 2 hr and change in rate ofextension calculated as initial--final rates.

FIG. 6. The effects of cucumber expansins on cellulose paper.

(A) Strips of Whatman No. 3 filter paper were clamped in a constant loadextensometer with 5 mm of paper between the clamps (described in FIG.1). Paper strips were initially bathed in 50 mM sodium acetate, pH 4.5.After about 20 min. of extension, the bathing solution was replaced witheither: 5 mg of S1 protein in 15 mM Mes, pH adjusted to 4.5 with 1 Macetic acid (S1); 5 mg of S1 protein, in the same buffer, which had beenheated to 100° C. for two minutes (Boiled S1); 100 mg of cellulase (fromTrichoderma viride Boehringer Mannheim) in 50 mM sodium acetate(Cellulase), pH 4.5; or 50 mM sodium acetate pH 4.5 (Control).Extensions were recorded for a further 100 min. The figure showsrepresentative traces from at least six independent experiments, all ofwhich showed similar results.

(B) Strips of Whatman No. 3 paper were soaked in a solution containing 5mg /mL S1 protein in 15 mM Mes, pH adjusted to 4.5 with 1 M acetic acid,(S1); in the same protein solution which had been boiled for two minutes(Boiled S1); or in 50 mM sodium acetate, pH 4.5 (Control). Stressrelaxation tests were performed by standard methods. Data represent theaverage of ten measurements in each case. Experiments were repeatedthree times with similar results.

(C) Conditions were the same as in (B) except that the paper strips weresoaked in a solution containing 100 mg/mL of Trichodenna cellulase in 50mM sodium acetate, pH 4.5 (Cellulase), or 50 mM sodium acetate, pH 4.5(Control). Data are the averages of ten measurements in both cases.Experiments were repeated three times with similar results.

FIG. 7 The effect of cucumber expansins on slick paper.

Paper strips of 2-3 mm width were cut and hung in our extensometer, suchthat a 5-mm long strip was put under 20-g force. The buffer contained 50mM sodium acetate, pH 4.5 with or without added C3 protein (about 20 μgper paper strip was used). The length of the paper strips was recordedfor up to 5 h. Four samples of each paper were used with each treatment(+/- protein). The untreated (control) paper showed very little changein length, and maintained its mechanical strength throughout the testperiod. In contrast, the treated papers began to extend and quicklybroke once extension began. The time for breakage after addition ofprotein varied from 45 min. to 2.5 h, but all samples broke.

FIG. 8. Acid-induced extension in native and heat-treated oat coleoptilewalls.

(A) When switched from a neutral buffer (50 mM Hepes, pH 6.8) to anacidic buffer (50 mM sodium acetate, pH 4.5), native walls extended at ahigh rate which fell continuously with time, whereas heat-treated wallslacked a significant response. The native and heat-treated (15 sec inboiling water) walls were prepared and mounted on an extensometer (5 mmbetween two clamps) with 20 g of tension as described by Cosgrove (1989)and McQueen-Mason et al. (1992).

(B) A plot of extension rate for the native walls shown in Ademonstrates that the rate decreases continuously. Similar results wereobtained in 10 repetitions.

FIG. 9. Effect of dithiothreitol (DTT) addition to acid-inducedextension of oat coleoptile walls.

Native walls were prepared as in FIG. 8 and the buffer was exchanged forthe same buffer containing 10 mM DTT at approximately 80 min after startof acid-induced extension

(A). The plot of extension rate (B) shows that the rate is stable atabout 5% per h after addition of DTT. Similar results were obtained in 6independent trials.

FIG. 10. Acid-induced extension in native oat coleoptile walls in thecontinuous presence of DTT.

(A) Walls were prepared as in FIG. 8 and incubated in 50 mM Hepes, pH6.8, with 2 mM DTT; after about 35 min the buffer was switched to 50 mMsodium acetate, pH 4.5, with 2 mM DTT.

(B) The plot of extension rate shows that the decay in extension rateafter 1 h is greatly diminished (compare with FIG. 8). This experimentwas repeated four times with similar results.

FIG. 11. Extension induced in oat, cucumber and pea walls by proteinsextracted from oat coleoptiles walls (A) and cucumber walls (B).

Coleoptile wall proteins and cucumber wall proteins were extracted with1 M NaCl, precipitated with ammonium sulfate, desalted and partiallypurified on a DEAE Sephadex column. Heat-inactivated cell walls from oatcoleoptiles, cucumber hypocotyls or pea epicotyls were clamped at 20-gtension in 50 mM sodium acetate, pH 4.5. After about 0.5 h, the solutionwas replaced with 0.4 mL of the same buffer containing about 10 mgproteins partially purified from oat or cucumber cell walls byDEAE-anion exchange chromatography. The data represent a typicalexperiment from at least four replicates.

FIG. 12. Comparative responsiveness of oat and cucumber walls toexogenously added oat or cucumber proteins.

(A) Concentration dependence of cucumber and oat walls to exogenouscucumber protein. Cucumber walls were frozen, thawed, abraded,heat-inactivated and loaded in the extensometer, as in FIG. 8. Oatcoleoptiles were treated similarly, except that the epidermis wasstripped with fine forceps and the coleoptile bisected longitudinallyprior to freezing. The walls extended for approximately 30 min in 50 mMsodium acetate, pH 4.5, before the buffer was exchanged for the same onecontaining various concentrations of partially purified cucumberexpansins (the active fraction from the C3 HPLC separation) . Extensionactivity is calculated as the increase in extension rate in the first15-20 min after addition of C3 protein and is expressed as % increase inlength per h). The average extension rate prior to addition of proteinin these experiments was 2% per h for oat walls and 1.7% per h forcucumber walls.

(B) Response of oat and cucumber walls to added oat protein.Heat-inactivated oat and cucumber walls were prepared as in (A) andextended in sodium acetate, pH 4.5, for about 30 min prior to exchangeof buffer for the same one containing 73 , μg of crude oat coleoptileprotein (ammonium sulfate precipitate) per sample holder (about 400 μL).The extension curves shown illustrate two examples with responses closeto the mean (2.8% h⁻¹ increase for the cucumber wall and 1.1% h⁻¹increase for the oat wall). The mean responses (n=4) of oat and cucumberwalls to added oat protein are shown in the inset and are calculated asthe increase in extension rate, expressed as % per h. The baselineextension rates prior to addition of protein averaged 2.4% per h (oats)and 1.1% per h (cucumber).

FIG. 13. Effect of 1 M NaCl extraction on acid-induced extension of oatcoleoptile walls.

Coleoptiles were abraded prior to freezing, then either directly clampedin the extensometer or extracted for 16 h in 1 M NaCl containing 10 mMHepes, pH 6.8, 3 mM EDTA and 2 mM sodium bisulfite prior to measurement.Walls were rinsed briefly in water, clamped at 20 g tension, thenincubated in 50 mM Hepes, pH 6.8, with 2 mM DTT. At about 30 min thebuffer was exchanged for 50 mM sodium acetate, pH 4.5, with 2 mM DTT.The curves shown are representative of eight trials for each treatment.The inset shows the average extension response (SE, n=8) of eachtreatment. Response is calculated as the rate at 1.5 to 2 h minus therate prior to pH 4.5 buffer, and is expressed as % increase in lengthper h.

FIG. 14. pH dependence of the extension activity reconstituted by oatwall proteins.

Heat-inactivated walls from cucumber hypocotyls were clamped in anextensometer as in FIG. 8 and placed in 50 mM sodium acetate buffers atpH 3.5, 4.0, 4.5, 5.0 or 5.5. After 20 min, the solutions were replacedwith 0.4 mL of the corresponding buffer containing 10 mg oat-coleoptilewall proteins partially purified by DEAE-chromatography. The extensionactivity was calculated by subtracting the baseline rate withoutproteins from the linear rate (5-30 min) after the addition of theproteins, and expressed as % increase in length per h above the baselinerate. The data represent the means ±S.E. (n=5 to 8). The averagebaseline rate prior to addition of protein ranged between 2.52 and 2.96μm min⁻¹ (or 3.0 to 3.55 % per h) for all pH groups.

FIG. 15. Purification of oat expansin by high pressure liquidchromatography (HPLC) on a carboxymethyl (CM) cation exchange column.

(A) Elution of proteins (absorbance at 280 nm) from a CM-column.Proteins were solubilized from the cell walls of etiolated coleoptilesby 1 M NaCl and then sequentially fractionated by ammonium sulfateprecipitation, DEAE-chromatography, and ConA chromatography, prior toCM-HPLC.

B) Wall extension activity of HPLC fractions. The extension activity wasassayed by addition of fraction samples (equal volumes, typically 20 μL)to sample cuvettes containing 400 μL of 50 mM sodium acetate buffer, pH4.5. Activity is expressed as increase in extension rate after additionof protein to a 5-mm, heat-inactivated, abraded oat coleoptile. A singlepeak of activity eluted at about 15 min or 3.5% of 1 M NaCl. Similarresults were obtained in six independent trials.

FIG. 16. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteinsfrom different purification steps.

Active protein fractions were separated on a SDS-PAGE gradient gel(4-20%) and stained with Coomassie Blue R-250. Lane 1, 1 M NaClextraction (20 mg); lane 2, ammonium sulfate precipitate (20 mg); lane3, active DEAE-fraction (10 mg); lane 4, proteins passing through Con Acolumn (5 mg); lane 5, active fraction from CM-HPLC (0.3 mg). Theprotein with apparent molecular mass of about 29 kD was designated asoat expansin (oat Ex29). Similar results were obtained in five trials,except in some of these an additional protein band at about 35 kDappeared in lane 5.

FIG. 17. Western analysis of oat coleoptile proteins probed withantiserum against cucumber Ex29.

Comparison of lane 1 (crude wall protein) with lane 2 (crudeprotoplasmic protein) shows that the antiserum recognizes an Ex29-likeprotein (arrow) specifically bound to the coleoptile cell wall.Comparison of lane 3 (crude wall protein) with lane 4 (purified proteinwith extension activity) shows that the Ex29-like protein co-purifieswith the extension activity. Methods: Lane 1 was loaded with 15 μg ofcrude wall protein (ammonium sulfae precipitate of 1 M NaCl extract).Lane 2 had 15 μg of the soluble protoplasmic protein fraction. Thesesamples were separated on the same 4-20% gradient SDS polyacrylamidegel, blotted onto nitrocellulose, and probed with rabbit antiserumagainst cucumber Ex29. Lane 3 was loaded with 0.2 μg of crude wallprotein and Lane 4 had 0.2 μg of active oat wall-extension proteinpurified sequentially by DEAE Sephadex and CM-HPLC. These samples (3-4)were separated on the same 14% SDS polyacrylamide gel, blotted ontonitrocellulose, and probed with antiserum. This assay, with minorvariations, was carried out four times with similar results.

FIG. 18. Extension induced in isolated cucumber walls by proteins fromsnails. Cucumber walls were prepared as in the description for FIG. 11.Proteins prepared by dissolving snail acetone powder ( 10 mg /mL) in 50mM sodium acetate, pH 4.5, is capable of inducing extension of isolatedcucumber walls. The immediate response and the linearity of theextension are unique characteristics that indicate an expansin--likeprotein may be involved.

FIG. 19. Extension induced in isolated cucumber walls by proteins fromsnails feces.

The feces of Helix aspersa (snail) induced extension in cucumber walls.The feces were dissolved in 50 mM sodium acetate buffer, pH 4.5. Theprocedure was as described in FIG. 11.

FIG. 20. Western blot analysis of active HPLC- separated proteinsfraction from snail.

Acetone powder solution was probed with antibody which was raisedagainst cucumber expansin -29. The analysis was performed as describedin FIG. 17. The active fractions show a striking band at about 26 kD,which is similar to cucumber expansin-29. These results provide strongevidence that the wall extension activity found in the snail acetonepowder is due to a protein of similar size and similar antigenicdeterminants as cucumber expansin-29.

FIG. 21. Western blot of the protein from Helix aspersa feces, probedwith the antibody PA-1.

Feces from Helix aspersa were dissolved as described in FIG. 19 andwestern blot analysis was performed as in FIG. 20. The single bandindicates the presence of an protein antigenically similar to cucumberexpansin-29.

FIG. 22. Effects of 2 M urea.

This figure shows the effects of 2 M urea on extension of native walls,walls reconstituted with expansin, and heat inactivated walls asdescribed in Example 16.

FIG. 23. Use of expansins to enhance the accessibility of cellulose tocellulase and related enzymes and processes.

This figure shows that the cellulose degradation curve of cellulase andexpansin is very different from that of cellulase only, or of expansinonly, and they do not have a superimposed relationship. It also showsthat cellulase and expansin have a synergistic effect in degrading ormodifying cellulose.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a new class of proteins named expansins.During the course of detailed studies of plant cell growth, ourlaboratory isolated first from cucumber two novel proteins which wenamed expansins. Further research found expansins in oat coleoptile aswell as in the proteins obtained from the digestive track of snails. Thesupport for these new findings and ways of using it commercially are theobject of the present invention.

A detailed embodiment of the present invention involving cucumberproteins cEx-29 and cEx-30, as well as oat protein oEx-29 andexpansin-like protein from snail digestive track and its feces sEx isherein disclosed. However it is understood that the preferred embodimentis merely illustrative of the invention which may be embodied in variousforms and applications accordingly, specific structural and functionaldetails disclosed herein are not to be interpreted as limiting, butmerely as a support for the invention as claimed and as appropriaterepresentation for the teaching of one skilled in the art to variouslyemploy the present invention in any appropriate embodiment.

The following abbreviations have been used in the specification:ConA=Concanavalin A; CM=carboxymethyl; DEAE=diethylaminoethyl;Hepes=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;DTT=dithiothreitol; Ex29=29-kD expansin; Mes=2-(N-morpholino)ethanesulphonic acid; HPLC=high-pressure liquid chromatography;PBST=phosphate buffered saline with Tween-20; SDS-PAGE--sodium dodecylsulfate-polyacrylamide gel electrophoresis;

Our basic approach was to solubilize ionically bound proteins fromgrowing cell walls and assay their ability to restore the endogenousextension activity in heat-inactivated walls. Extension was assayedusing a constant load extensometer, in which tissue samples were clampedunder constant force and their extension recorded using an electronicdisplacement transducer (Cosgrove, D. J. Planta, 177:121-130, 1989). Thefirst two proteins possessing the ability to restore the extensionactivity in heat- inactivated wall were solubilized from wall fragmentsisolated from the growing hypocotyls of dark-grown cucumber seedlings.After various unsuccessful attempts at reconstitution, we obtained acrude salt-solubilized fraction with the ability to induce extension ininactivated walls, shown in FIG. 1A. The extension induced by thisextract mimicked the native extension activity in magnitude andkinetics, i.e. initially high rates decayed over a period of 2 hr tomore stable rates of about 2% per hour. These rates are lower thanelongation rates of the living stem, but the stress applied to theisolated walls was only one-fifth of the equivalent longitudinal stressimposed on the walls by cell turgor. Like the endogenous extensionactivity of isolated cell walls, the reconstituted activity required anacid pH and was irreversible (i.e. upon removal of load, the segmentsdid not return to their original length). The addition of protein tonative cell walls did not substantially enhance extension, suggestingthat endogenous activity was saturating or that binding sites were notaccessible to the added material.

The active material appeared to be restricted to the growing region ofthe hypocotyl. When wall fragments from basal (non growing) stem tissueor from the cotyledons (which expand negligibly in our conditions) wereextracted, proteins solubilized in the same way did not induce wallextension (FIG. 1A). These results suggest that nongrowing tissues lackthe active material; however, we cannot exclude the possibility that theactive material was present in the tissue but more firmly bound,inactivated during extraction, or lost during wall isolation. Solublecytoplasmic proteins (collected as expressed cell sap or as homogenate)showed no activity, whilst proteins which were salt solubilized in anequal volume from cleaned wall residues taken from the same tissueshowed clear extension inducing activity, indicating an association ofthe extension activity with the wall.

The wall extract from growing cucumber walls did not induce extension ofwalls from the basal (non elongating) stem (FIG. 1A). Evidently, duringmaturation the wall is biochemically modified so that it is notsusceptible to extension by this material. Perhaps peroxidativecross-linking of lignin or structural proteins such as extensin isinvolved in this loss of sensitivity.

The extension activity showed an interesting pattern of speciesspecificity. FIG. 1B shows that the cucumber wall extract was active onvarious dicot seedlings (pea, radish, cucumber and tomato) and onmonocots of the Amaryllidaceae (onion and zephyr lily). In contrast, theextract had a much smaller effect on the coleoptile wall of graminaceousmonocots (maize and barley). As graminaceous monocot cell walls differfrom those of dicots by having less pectin and hydroxyproline-richglycoprotein, and also by having a different type of hemicellulose, itmay be that the active fraction extracted from cucumber walls interactswith one or more of these components to induce extension. It may also bethat these grass cell walls are cross-linked (not necessarilycovalently) in a manner that renders them immune to the cucumberextract. In contrast monocots of the Amaryllidaceae have a cell wallcomposition more similar to dicots than to the graminae, and thisprobably explains their susceptibility to the cucumber derived activity.

The cucumber wall extract was separated by ammonium sulfateprecipitation followed by sequential HPLC, shown in FIG. 2, first usinga hydrophobic interactions column, where a single peak of activitydesiphated as the C3 proteins was obtained, and then using a cationexchange column from which the activity was eluted as two distinct peaksThese fractions we have designated S1 and S2 with respect to their orderof elution. FIG. 3 shows the analysis of the active fractions bySDS-PAGE which revealed a major band of relative molecular mass 29 kDassociated with S1, while S2 contained a major band at 30 kD (FIG. 3).Active extracts have also been separated by native PAGE and by liquidchromatography with hydroxyapatite, gel filtration media, and DEAE anionexchangers, where activity was consistently associated with these twobands (data not shown). The S1 fraction required only 0.3-1.0 microgramsof protein to reconstitute extension rates similar to that of nativeextension, while it required 1.0-2.0 micrograms of S2 to do this.

Cucumber expansins induced extension of walls from several dicot andmonocot species, but had little effect when assayed with coleoptilewalls from maize and barley. Grass coleoptiles have been a favoredobject of growth studies since Darwin's analysis of phototropism andhave been instrumental in many discoveries about plant growth, includingthe discovery of auxin. The wall composition of grass coleoptiles isnotably different from that of dicots and other monocots nevertheless,coleoptile walls, like dicot walls, do exhibit a strong acid-inducedextension in vitro and in vivo. These observations led us to postulatethat a wall protein, with functions analogous to the cucumber expansins,might mediate the endogenous acid-induced extension of coleoptile walls.Therefore, in further study we attempted to identify oat coleoptileproteins that could induce wall extension, using the wall extraction andreconstitution approach that proved successful with cucumber walls.

To identify the hypothetical oat proteins responsible for wallextension, we used 1 M NaCl to extract proteins from cell walls ofetiolated oat coleoptiles. Proteins in this crude extract wereprecipitated with ammonium sulfate, desalted, and fractionated on a DEAESephadex anion exchange column. The unbound proteins passing throughthis column possessed the ability to induce extension inheat-inactivated walls from oat coleoptiles (FIG. 11A). Moreover, thisactive fraction also induced extension of heat-inactivated cucumberhypocotyl walls and pea epicotyl walls (FIG. 11A). This result wassurprising because earlier results led us to expect poorcross-reactivity between extension proteins and walls from dicots andgrass coleoptiles.

FIG. 8 shows an example of acid-induced extension of oat coleoptilewalls. When native walls were clamped under constant tension at neutralpH, the extension rate was low 30 min after application of the load andcould be greatly increased by replacing the neutral buffer with a bufferof pH 4.5. The wall extension rate decreased continuously with timeafter the change to acid buffer (FIG. 8B). Addition of 10 mMdithiothreitol, a sulfhydryl reducing agent, increased the extensionrate and stabilized it (FIG. 9). This effect is similar to thatpreviously found with cucumber walls (Cosgrove 1989). Inclusion ofdithiothreitol throughout the extension period resulted in a simplerdecay to a constant extension rate (FIG. 10). Pretreatment of thecoleoptile wall with boiling water for 15 sec to inactivate wallproteins eliminated the acid-induced extension (FIG. 8A), suggestingthat extension of these walls may be due to a protein-mediated reaction.These observations are consistent with previous reports of acid-inducedextension in oat and cucumber walls.

To pursue this point further, we tested the ability of cucumberexpansins to induce extension of oat coleoptile walls. As shown in FIG.11B, they indeed induced extension in oat coleoptile walls, but withless effectiveness than when assayed with cucumber walls. This lastpoint is quantified in FIG. 12A, which compares responsiveness ofcucumber and oat walls to a partially purified cucumber expansinfraction. The maximal response of cucumber walls was about three timeshigher than that of oat walls. To obtain the same extension response inoat walls as was elicited in cucumber walls by 2 μg mL⁻¹ of cucumberprotein, ten times as much protein was required. FIG. 12B shows thatcucumber walls were similarly more sensitive to the active oat extractthan were oat walls. The protein-induced extension was stabilized byinclusion of 1-10 mM dithiothreitol in the incubation buffer (data notshown). We also confirmed that cucumber expansins caused very littleextension of barley coleoptiles (data not shown). Thus, we conclude thatoat coleoptile walls are substantially more responsive in thesereconstitution assays than barley coleoptile walls, but less responsivethan cucumber walls.

Despite the quantitative differences, these results show that oat andcucumber proteins can induce qualitatively similar extension responsesin oat and cucumber walls. We thus infer that a similar biochemicalmechanism is involved in acid-induced wall expansion in grasses as indicots.

One might expect that extraction of frozen/thawed coleoptiles with 1 MNaCl might remove most or all of the acid-extension response. FIG. 13shows that overnight extraction of coleoptiles indeed diminished theiracid-extension response. This loss could be due to extraction of theexpansins; alternatively, the walls or their proteins may have beenmodified in some other way so that they lost the acid-extensionresponse. Cucumber walls were previously found to lose their nativeextension response when they were pre-incubated in neutral pH (Cosgrove1989). Further work will be needed to differentiate between these andother possible explanations.

To assay the pH dependence of the extractable expansin activity fromoats, we used cucumber hypocotyl walls as the "substrate" for measuringthe extension activity of oat proteins. Cucumber walls were used becausethey were easier to prepare, broke less often, had lower baselineextension rates, and proved to be a more sensitive substrate forextension assays than did the walls from oat coleoptiles. The active oatfractions from the DEAE-column had a pH optimum between 4.5 and 5.0(FIG. 14). At pH 3.5 or 5.5, the activity was reduced by about 50%. Incontrast, cucumber expansins displayed a broader pH optimum, with highactivity maintained at pH 3.5.

When the active proteins from the DEAE-column were passed through aConcanavalin A (Con A) column, the majority of the extension activitydid not bind to the lectin column, suggesting that the activity was notassociated with glucosyl- or manosyl-glycoproteins. When the activefractions which passed through the Con A column were furtherfractionated by HPLC on a carboxymethyl cation exchange column,extension activity was eluted as a single major peak at about 15 min(FIG. 15). The activity of this protein fraction had an acid optimumsimilar to that shown in FIG. 14 (data not shown). SDS-polyacrylamidegel electrophoresis revealed that a protein with molecular mass of about29 kD was associated with this active fraction (FIG. 16). We designatethis protein as oat expansin-29 (oat Ex29, oEx-29).

A summary of the purification steps for the oat Ex29 is shown inTable 1. This protein was purified sequentially by ammonium sulfateprecipitation, DEAE-chromatography, Con A affinity chromatography and CMcation exchange chromatography. If the extension activity inammonium-sulfate-precipitated proteins is taken as 100%, a purificationof 51 fold with 69% yield was obtained after two steps of ion exchangechromatography. Because Con A affinity chromatography did notsignificantly purify oEx-29, this step was often omitted withoutsignificant effect on the purification.

To examine whether expansin activity was also present in the protoplasmof oat coleoptiles, we fractionated the soluble protoplasmic proteins(all proteins in the coleoptile homogenate not bound to the wall) byammonium sulfate precipitation and DEAE-column chromatography. Little orno activity was detected in any fractions (data not shown), suggestingthat the responsible protein was bound to the cell walls of oatcoleoptiles.

Because oat Ex29 was capable of catalyzing extension of walls fromcucumber, we wished to test whether it is immunologically related to the29-kD cucumber expansin cEx-29. FIG. 17 shows that the oat Ex-29 wasspecifically recognized by an antibody raised against cucumber Ex-29.Pre-immune serum under the same conditions did not label Ex-29 (notshown). Little signal was detected in the soluble protoplasmic fractionof oat L coleoptiles, which is consistent with our other evidence thatthe Ex-29 is a wall-bound protein. When equal amounts of crude andpurified wall protein were assayed by Western analysis, the purifiedprotein gave a much greater signal (FIG. 17). These results demonstratethat the extension-inducing activity co-purifies with the protein thatis antigenically related to cucumber Ex-29.

Our results show that oat coleoptile walls possess a protein that canmediate acid-induced extension of grass coleoptile walls and dicotwalls. This protein resembles the cucumber 29-kD expansin in that itinduces extension at acid pH but not at neutral pH, its activity isstabilized by dithiothreitol, it has a similar size as judged bySDS-PAGE, and it is specifically recognized by an antiserum raisedagainst the cucumber protein. Because of these similarities between theoat and cucumber expansins, we propose that expansins are evolutionarilyconserved proteins that underlie at least part of the acid-extensionresponse common to the walls of many plant species.

The similarity between oat and cucumber expansins surprised us becausewe found that cucumber expansins caused little extension of maizecoleoptile walls and no extension of barley coleoptile walls. Weconfirmed that barley walls were unresponsive to cucumber expansins, butour positive results with oat coleoptiles show that this insensitivityis not a general property of grass coleoptiles. We do not know whybarley walls failed to extend in the presence of cucumber or oatexpansins.

The similarity between oat and cucumber expansins is also surprisingbecause the matrix components of the wall are believed to be importantfor wall loosening and extension, yet these components are quitedifferent for grass walls and dicots. The major matrix polysaccharidesof the coleoptile wall are (1→3, 1→4)-β-D-glucans and arabinoxylanswhereas dicots contain principally xyloglucans and pectins. Dicot wallscontain hydroxyproline-rich glycoproteins of the extensin family whereasgrass walls contain much lower amounts of such proteins. It may be thatexpansins interact directly with wall components common to both types ofwalls (e.g. cellulose or a minor matrix component) or that they can acton different glycans with similar functions, e.g. xyloglucans and (1->3,1->4)-β-D-glucans.

Although oat Ex29 can induce an acid-dependent extension in coleoptilewalls, there are notable differences between the native andreconstituted acid-extension responses in coleoptiles. First, the acidresponse of native walls includes a large, but transient, burst inextension, which is mostly decayed away by 60-90 min. This burst islargely lacking in oEx29-reconstituted extensions, which resemble moreclosely the steady extensions which outlast the transient (e.g. FIG. 8and 10). Second, the pH dependence of reconstituted extension (FIG. 14)does not exactly match the pH dependence reported for acid-extensionresponses of isolated coleoptile walls. The reconstituted extensionsdisplayed a maximum at pH 4.5 and fell off at lower pH values, whereasacid extensions of native coleoptile walls did not fall off at lower pHvalues. Third, the maximum extension rate inducible with exogenous Ex29was substantially less than acid-extension responses of nativecoleoptile walls (i.e. about 2% h⁻¹ for reconstituted extensions versusabout 5% h⁻¹ for the stable component of native wall extensions or 20-30% h⁻¹ for the immediate response of native walls).

These differences between the reconstituted and native acid-extensionresponses of coleoptile walls could indicate that oat coleoptilespossess additional acid-extension processes, other than the one mediatedby oEx-29. On the other hand, these differences might also result frominadequacies in our reconstitution methods. For example, heatinactivation of the coleoptile wall by treatment with boiling water maymodify the wall's structure so that it is not as susceptible to oEx-29action. There may also be differences due to poor accessibility ofexogenous Ex-29 to its site of action in the wall, or with need forancillary wall enzymes that are inactivated by heat treatment. Hence, webelieve that, at this stage, caution is warranted in interpreting thedifferences between native and reconstituted extensions.

From the characteristics of oEx-29-induced extension of coleoptile wallsand the above considerations, we propose that oat Ex29 is responsiblefor at least part of the long-term (>1 h) acid-induced extensionresponses of oat coleoptiles. This view is strengthened by the findingsthat reconstituted wall extension and endogenous wall extension exhibitsimilar sensitivities to biochemical activators and inhibitors. Incucumber, exogenous expansins can restore extension rates as high orhigher than the long-term extension rates in native walls exposed toacid pH. Although we believe there to be good reasons for thinking thatexpansins mediate the long-term acid-induced extension of isolatedwalls, their role in the growth of living tissues has not been directlyaddressed. This assessment will require experiments in which the actionof expansins are specifically inhibited or enhanced. Further studies ofthe biochemical action of expansins and the genes that encode them mayprovide the tools for this assessment.

Digestive tracks of many animals contain enzymes able to catalyzereactions weakening various chemical bonds. We postulated that thedigestive track of animals which consume green parts of plants maycontain proteins of action similar to expansins. We found expansin-likeproteins in the digestive track and feces of the snail Helix aspersa.Western blot analysis of the snail proteins probed with the antibodyraised against cucumber expansins cEx-29 indicates the presence of anprotein antigenically similar to cucumber expansin.

The expansin-like proteins in the snail digestive track may besynthesized by the snails or by an endosymbiont living in the digestivesystem of the snail. Such symbionts might be bacteria, fungi, protistsor some other microbe.

We assayed the effects of expansins on the mechanical properties of purecellulose paper, which derives its strength principally from hydrogenbonding between cellulose microfibrils. Because paper is much simpler incomposition and structure than plant cell walls, the action of expansinscould be much easier to interpret. S1 protein showed clear effects onthe mechanical properties of Whatman number 3 filter paper in bothextensometer and stress relaxation assays. As our experiments showexpansins are also able to disrupt slick paper (FIG. 7).

Expansins appeared to weaken cellulose paper in a manner different fromthat effected by cellulase. While both expansins and cellulase led tobreakage of the paper in extensometer assays, only expansins showed aclear enhancement of stress relaxation in this material. Furthermore,incubations of paper with expansins did not lead to the release ofreducing sugars, as was seen in incubations with cellulase. Becauseexpansins did not hydrolyze the paper, and because hydrogen bondingaccounts for the tensile strength of paper we conclude that expansinsdisrupt the hydrogen bonding between cellulose fibrils. Expansinsprobably do not catalyze cell wall loosening by disrupting hydrogenbonds directly between microfibrils, as these structures appear to beseparated from one another by matrix polysaccharides which are thoughtto coat the outside of microfibrils in the wall. However, it is possiblethat they could induce wall extension by disrupting hydrogen bondingbetween the matrix polysaccharides and the microfibrils, thus reducingthe resistance to movement of the microfibrils.

Stress relaxation assays performed to test the ability of the snailexpansin-like protein to weaken the bonds in the paper indicate thatthis ability is attributed to all expansins and expansin-like proteins,

EXAMPLE 1

Preparation of Plant Materials

Seeds of cucumber (Cucumis sativus cv Burpee Pickler) were sown on SeedGermination Kimpack Paper K-22 (Seedburro Equipment Corporation,Chicago, Ill.) soaked with distilled water, in flats, 50×25×6 cm, withlids of the same dimensions. Seedlings were grown in the dark for 4 daysat 27° C. The apical 3 cm of hypocotyl was excised and frozen at -20° C.for no more than 5 days and prepared for creep measurements aspreviously described (Cosgrove 1989). For bulk wall extractions, theapical 3-cm hypocotyl regions were collected on ice water andhomogenized with 25 mM sodium acetate, 2 mM EDTA, pH 4.5 in a Waringblender. The wall fragments were collected and washed twice byfiltration through Miracloth and subsequently used for proteinextraction. Basal walls were from the lower 6 cm of the (15 cm long)hypocotyls.

Oat seedlings were grown in moist vermiculite in complete darkness at27° C. Except as noted, coleoptiles were from 4-day old oat seedlings(Avena sativus L. cv. Olge, from Carolina Biological Supply, Burlington,N.C., USA).

Seedlings were quickly harvested under room lights. For wall extensionassays, the apical 2 cm region of the growing stem or coleoptile wasexcised, sealed in aluminum foil and frozen at -20° C prior to use.Coleoptiles were separated from primary leaves. Cuticles were abraded byrubbing the coleoptiles or stems between two fingers coated with aslurry of carborundum (320 grit, well washed prior to use; FisherScientific, Fair Lawn, N.J.). For oat coleoptiles, the cuticle wasgenerally abraded prior to freezing, whereas for cucumber and pea stemsthe frozen segment was quickly abraded. In some instances as noted,coleoptile cuticles were removed by stripping the epidermis from thetissue with fine forceps and the remaining coleoptile cylinder wasbisected longitudinally prior to freezing. Tissues were thawed, pressedunder weight for 5-10 min to remove tissue fluids and clamped in anextensometer (5 mm between the clamps, corresponding to the apical 3-8mm of the stem or coleoptile).

Seeds of pea (Pisum sativum cv Alaska), radish (Raphanus sativus cvCrimson Giant), tomato (Lycopersicon esculentum cv Rutgers), onion(Alium cepa cv Snow White), maize (Zea mays cv B73×Mol17), and barley(Hordeum sativum cv Barsoy) were sown on vermiculite wetted withdistilled water and grown for 4 days in the dark at 27° C. Hypocotyls ofpea, radish and tomato; primary leaves of onion; and coleoptiles ofmaize and barley were excised and frozen for subsequent extensionexperiments. Young growing leaves of zephyr lily (Zephyranthes candida)were removed from a greenhouse-grown colony of plants which were kept inthe dark at 27° C. for 12 hr prior to harvest, and frozen for laterextension assay.

EXAMPLE 2

Isolation of Expansins cEx-29 and cEx-30 from Cucumber

Protein Extraction

Washed cucumber cell wall fragments (from 150-200 g of tissue) wereextracted overnight in 20 mM Hepes, pH 6.8, 1 M NaCl at 4° C. Cell wallfragments were filtered on Miracloth and the salt-solubilized fractionprecipitated with ammonium sulfate (the activity precipitated between 20and 60% saturation with [NH₄ ]₂ SO₄). The precipitate was desalted on a7-mL column of Bio-Gel P2 (Bio-Rad Laboratories) into 50 mM sodiumacetate, pH 4.5. Protein concentration was 2 to 4 mg/mL, estimated byCoomassie Protein Assay Reagent (Pierce, Rockford, Ill.).

For the comparison of soluble and wall associated proteins, 100 g oftissue was harvested and homogenized with 100 mL of 25 mM sodiumacetate, pH 4.5, 1 mM EDTA. Wall fragments were filtered out and theremaining solution designated as the soluble fraction. Wall fragmentswere cleaned as described above, and then extracted in 200 mL of 20 mMHepes, pH 6.8, 1 M NaCl for one hour at 4° C. Aliquots of both solutionswere dialyzed against 50 mM sodium acetate pH 4.5 and tested forreconstitution activity, as described below.

Protein Fractionation

The 20 to 60% (NH₄)₂ SO₄ precipitate pellet was resuspended in 2 mL ofdistilled water and insoluble material was removed by centrifugation andfiltration through a Centricon 30 microconcentrator (Amicon, Beverly,Mass.) prior to being loaded onto a C3 hydrophobic interactions column(ISCO C-3/6.5 μm 10×250 mm) equilibrated with 50 mM sodium acetate, pH4.5, 20% (saturation) (NH₄)₂ SO₄. Proteins were eluted from the columnwith a linear gradient from the equilibration buffer into 50 mM sodiumacetate, pH 4.5, in 35 min. at a flow rate of 1 mL/min. Fractions weredesalted on a 7-mL column of Bio-Gel P-2 (Bio-Rad Laboratories,Richmond, Calif.) into 50 MM sodium acetate and assayed for theirability to reconstitute extension to inactivated cucumber hypocotylsections.

The active fractions from the C3 column were pooled and subsequentlydesalted and concentrated on a Centricon 30 microconcentrator (Amicon,Beverly, Mass.), the buffer being exchanged for 15 mM Mes NaOH, pH 6.5.The concentrated sample was then loaded (in a volume of 1.7 mL) onto asulfopropyl cation exchange column (Bio-Rad HRLC MA7S 50×7.8 mm)equilibrated with 15 mM Mes NaOH, pH 6.5, and proteins eluted with anascending gradient of NaCI (from 0 to 1.0 M over 45 min) in the samebuffer at a flow rate of 1 mL/min. A₂₈₀ nm of eluting proteins wasmeasured using a Dionex Variable Wavelength Detector (VDM-2).

Active fractions from ammonium sulfate precipitation, C3 column andsulfopropyl fractions S₁ and S₂ were concentrated, desalted on Centricon30 Microconcentrators, and then run on SDS-polyacrylamide gels,according to the method of Laemmli. Gels were subsequently stained withCoomassie Brilliant Blue R250. Fractionation by this procedure wasrepeated more than 10 times with similar results. Results are presentedin FIG. 2.

EXAMPLE 3

Isolation of Expansins oEx-29 from Oat

Protein Extraction

For oat protein extraction, oat seedlings were rapidly cut under roomlights and placed in ice water. The apical 2.5 cm (+/- 0.5 cm) of eachcoleoptile was then cut, separated from the primary leaf, and placed onice while the other coleoptiles were harvested. About 500 coleoptileswere homogenized in 200 mL of 10 mM sodium phosphate, pH 6.0. In someinstances the coleoptiles were collected in lots of 100-200 and frozen(-20° C.) for 1 to 3 d prior to homogenization. The homogenate wasfiltered through a nylon screen (70 mm mesh), and the cell walls werecollected and washed 4 times by resuspending in the homogenizationbuffer (300 mL) followed by filtration. Ionically-bound proteins wereextracted for at least 1 hour at 4° C. with 50 mL of 1 M NaCI containing20 mM Hepes (pH 6.8), 2 mM EDTA and 3 mM sodium metabisulfite. Wallfragments were removed by filtration or centrifugation and the wallproteins in the supernatant were precipitated with ammonium sulfate (0.4g added to each mL). Precipitated proteins were dissolved in 1.5 mL ofwater and desalted on an Econo-Pac 10 DG desalting column (BIO-RADLaboratories, Richmond, Calif., USA) which was equilibrated with 20 mMTris-HCl, pH 8.0, containing 100 mM NaCl. Without NaCl the activeproteins tended to bind to the desalting column, resulting in a lowerrecovery.

Protein Fractionation

Protein solution from the desalting column was centrifuged in amicrocentrifuge for 3 min to remove precipitates. Proteins were thenloaded onto a DEAE-column (Sephadex A-25, Sigma) equilibrated with 20 mMTris-HCl, pH 8.0, containing 100 mM NaCl at 25° C. The proteins bound tothe DEAE-column were eluted by 1 M NaCI in 20 mM Tris-HCl buffer, pH8.0.

A 1-mL Concanavalin A column (Sigma) was equilibrated with 200 mM NaClcontaining 1 mM each of Mg⁺², Ca⁺², and Mn⁺². Proteins from theDEAE-column were passed through this column. After the column was washedextensively with the same solution, the bound proteins were eluted withthe same solution containing 0.5 M a-methyl manoside. Extension activitywas associated with the fractions that did not bind to the column.

Active fractions were further separated by HPLC using a carboxymethyl(CM) cation exchange column (4.6×250 mm, CM300/6.5 mm, ISCO, Nebr.)equilibrated with 10 mM Mes, pH 5.5. Before the protein sample (1 mL)was loaded on the column, the sample buffer was exchanged for 10 mM MES,pH 5.5, by use of a 30-kD Centricon microconcentrator (Amicon, Berverly,Mass., USA). Proteins were eluted from the CM column at a flow rate of 1mL/min with a gradient of 0 to 0%, 0 to 4%, 4 to 6% and 6 to 100% of 1.0M NaCl in 10 mM MES, pH 5.5, from 0 to 5 min, 5 to 10 min, 10 to 30 minand 30 to 50 min, respectively, and detected by absorbance at 280 nm.

Proteins were quantified colorimetrically using the Coomasie ProteinAssay Reagent (Pierce, Rockford, Ill., USA). For SDS-PAGE (Laemmli1970), proteins were separated on a 14% polyacrylamide gel or a 4-20%gradient polyacrylamide gel (Bio-Rad Ready Gel, Richmond, Calif., USA).For Western analysis, proteins were electrophoretically transferred to anitrocellulose membrane in a solution of 192 mM glycine, 25 mM Tris, 20%methanol (v/v) at 10 volt/cm for 3 h or in some cases 16 h. After themembrane was blocked with 3% bovine serum albumin in phosphate bufferedsaline containing 0.05% Tween-20 (PBST), it was incubated for 2 hr inPBST containing antiserum (1:3000 dilution). The membrane was washed 4times with PBST and then incubated for 1 h with goat anti-rabbitIgG-conjugated alkaline phosphatase (Sigma; dilution of 1:4000) in PBST.The Western blot was developed using bromochloroindolyl phosphate/nitroblue tetrazolium and the reaction was stopped with 10 mM EDTA.

Table I. Purification of oat Ex29 from etiolated oat coleoptiles.Activity was assayed as described in FIG. 4 and expressed as the initialincrease in the extension rate of isolated cucumber cell walls uponaddition of the protein fraction (e.g. 5 to 30 min after proteinaddition). Total activity was calculated by dividing the activity(measured in 1-3 extension assays) by the fraction of protein used foreach assay. Specific activity was calculated by dividing the totalactivity by the total protein.

    ______________________________________                                                          Total    Specific                                                     Total   activity activity                                                     Protein units    (units/mg                                                                            Purification                                                                          Yield                               Purification step                                                                       (μg) (μm/min)                                                                            protein)                                                                             (fold)  (%)                                 ______________________________________                                        (NH.sub.4).sub.2 SO.sub.4                                                               444     123      278    1       100                                 precipitation                                                                 DEAE      190     104      546    2       84                                  CM-HPLC    6       85      14278  51      69                                  ______________________________________                                    

Soluble protoplasmic proteins were obtained by homogenizing oatcoleoptiles in 10 mM sodium phosphate buffer (in some cases, 10 mMHepes, pH 6.8) and centrifuging at 4° C. at 26,000 g for 10 min toremove particulate matter.

EXAMPLE 4

Preparation of Expansin-like Proteins from Snails and Snail Feces

Acetone powders of the visceral humps of Helix pometia were purchasedfrom Sigma Chemical Company. Live snails (Helix aspersa) were purchasedfrom NASCO. Protein solution was made by dissolving snail acetone powder(10 mg/ml) in 50 mM sodium acetate buffer, pH 4.5. Feces of Helixaspersa were dissolved in the same buffer.

EXAMPLE 5

Extension Measurements

Extension measurements were performed using a constant load extensometeras described by Cosgrove (1989). Briefly, frozen/thawed tissues wereabraded for a short time with carborundum to disrupt the cuticle, boiledin water for 15 sec (for reconstitution assays), and secured between twoclamps (with about 5 mm between the clamps) under a constant tension of20 g force (for assays with radish, tomato and onion tissues the tensionwas reduced to 10 g). Plastic cuvettes were fitted around the walls andfilled with bathing solution. For reconstitution assays the bathingsolution was first 50 mM sodium-acetate pH 4.5 for 30 min., followed bythe protein fraction to be assayed in the same, or equivalent pH buffer.Movement of the lower clamp was detected with a position transducer andrecorded on a microcomputer. All extension assays reported here wererepeated at least five times, except for the heterologousreconstitutions (e.g. with walls from pea, tomato and corn), which wereperformed three to five times.

Cucumber Expansins Activity

Extension induced by cucumber expansins is presented in FIG. 1, 11, 12.

Oat Expansins Activity

Extension induced by oat expansin is presented in FIG. 11 and 12.

Snails Expansins Activity

Extension induced by snail expansin-like proteins are presented in FIG.18 and 19. The experiment was performed as described for cucumber andoat proteins.

The protein solution obtained from snail acetone powder by dissolvingsnail acetone powder (10 mg /mL) into 50 mM sodium acetate, pH 4.5, iscapable of inducing extension of isolated cucumber walls.

EXAMPLE 6

Effects of DTT and Metal Ions on Endogenous Acid Induced Wall Extension

Endogenous acid-induced extension was previously shown to be sensitiveto a number of exogenously applied factors (Cosgrove D. J., Planta vol.177, pp 121-130, 1989). The thiol reducing compound dithiothreitol (DTT)was shown to stabilize or even enhance the extension of isolatedcucumber cell walls at acid pH. To see if this was a characteristic ofour extension inducing proteins, we first reconstituted wall specimenswith an active C3 fraction from cucumber, as shown in FIG. 4a, stemswere allowed to extend for one hour at which point sufficient DTT wasapplied to bring the bathing solution to 10 mM. Almost immediately therate of extension is increased and thereafter remained constant, whilstreconstituted sections without the addition of DTT typically show agradual decline in the rate of extension.

In contrast heavy metal ions including copper and aluminum were shown tobe strongly inhibitory to endogenous acid-induced extension (Cosgrove1989). Again, in FIG. 4a, boiled stem tissue reconstituted with C3proteins showed very clearly the same sensitivity to an application ofthese ions, the rates of extension falling rapidly to the rate seen inthe inactivated tissue before reconstitution.

EXAMPLE 7

Effects of Boiling in Methanol or Water on the Activity of CucumberExpansins

An unusual property of the endogenous cell wall extension activity isits ability to survive boiling in methanol (Cosgrove, 1989). We foundthat methanol-boiled walls retained extractable activity, whereasextracts from cucumber tissues boiled in water do not exhibit thisactivity, shown in FIG. 4a. Fractionation of the active extract frommethanol boiled walls by the methods given above showed it to containthe 29- and 30-kD proteins in the S1 and S2 fractions (data not shown).The ability of the activity to survive boiling methanol may be due tothe small size of these proteins and to protection within theircarbohydrate-rich sites within the cell wall.

Protein concentrations were estimated using Coomassie Protein AssayReagent (Pierce, Rockford Ill.) using a standard curve constructed usingBSA (Pierce). Wall specimens inactivated by boiling were preincubatedwith S1 and S2 proteins at estimated concentrations of 5 micrograms permL and then extended in the extensiometer, as described above, in 50 mMsodium acetate pH 4.5. After 1 hr enough dithiothreitol (DTT), from a100 mM stock, was added to bring the bathing solution to 10 mM DTT.

To study the effect of heavy metal ions wall specimens were extendedinitially in 50 mM sodium acetate, pH 4.5. After 20 minutes the bathingsolution was exchanged for one containing an estimated 50 micrograms ofactive C3 proteins. One hour after the start of the experiment AlCl₃.6H₂O or CuCl₂.2H₂ O, from 100 mM stocks , were supplied to give a finalconcentration of 1 mM.

For both treatments 100 g (fresh weight) of apical tissue was harvestedand either boiled for 30 seconds in 1 L of distilled water, or for threeminutes in 1 L of Methanol. Methanol boiled tissue was allowed to drybefore being resuspended in 50 mM sodium acetate, pH 4.5. Both sets oftissue were then homogenized, cleaned and salt extracted, as describedabove. Salt extracts were precipitated with (NH₄)₂ SO₄, resuspended anddesalted, and tested for reconstitution activity as described above.

EXAMPLE 8

Effect of Protease Treatments on the Activity of Cucumber Expansins

Cosgrove (1989) also showed that endogenous wall extension waseliminated by incubation with proteases, one of the initial clues thatextension was catalyzed by proteins. The proteinaceous nature of ourextension inducing factors was indicated by the sensitivity ofreconstituted extension to protease incubations. This data is presentedin FIG. 4b; 4-hr incubations with chymotrypsin, papain, pronase andtrypsin all reduced extension to rates seen in inactivated tissues.

Inactivated stem sections were incubated with active C3 proteins, at anestimated concentration of 50 micrograms per mL, for 30 min at 25° C.Reconstituted stems were then incubated with 1000 units of trypsin or1000 units of chymotrypsin in 50 mM Hepes-NaOH, pH 7.3 for 4 hr at 30°C., and with pronase (2 mg/mL), or papain (2 mg/mL) in 50 mM sodiumacetate, pH 5.0, 1 mM DTT for 4 hr at 30° C. For a control C3 proteinswere incubated at 30° C. for 4 hr without the addition of proteases.After incubation the stem sections were assayed for extension, initiallyin 50 mM Hepes, pH 6.8, after 30 min the buffer was switched to 50 mMsodium acetate, pH 4.5. The difference in the rates of extension (pH4.5-pH 5.5) was calculated. All experiments were repeated four times.

EXAMPLE 9

Effects of pH on Reconstituted Extension Induced by Cucumber Expansins

Reconstitution experiments were carried out at arrange of values of pH,results presented in FIG. 5 show that there is little or no induction ofwall extension by the C3 proteins until between pH 5.5 and pH 5.0 andthat extension activity has a maximum some where between pH 4.5 and pH3.5. The region of highest response to changes in pH lies in the rangepH 5.5 and pH 4.5, these values are within the range of apoplastic pHvalues reported in elongating tissues, and supports the possibility thatthese proteins could play an important role in the acid-growth response.

Buffers in the range pH 3.0 to pH 6.5 were prepared by titrating 50 mMcitric acid with 1 M K₂ HPO₄ Inactivated stem sections were suspended inthe extensiometer in buffer of appropriate pH. After 30 min of extensionthe bathing solution was replaced by a 1:1 dilution of C3 proteins(estimated final concentration 50 micrograms per mL) with theappropriate buffer, where necessary the pH was adjusted using either 1 Mcitric acid or 1 M K₂ HPO₄. Change in rate of extension was calculatedas the difference in extension rates before and after addition ofproteins. All experiments were repeated eight times.

EXAMPLE 10

Effects of Cucumber Expansins on Cellulose Filter Paper

Strips of Whatman number 3 filter paper (Whatman Lab sales, HillsboroOR) (2 mm by 10 mm) were cut and were clamped in the constant loadextensometer as described for cucumber hypocotyl sections. Extension wasmeasured in 50 mM sodium acetate and in the same buffer containingprotein fractions. Additionally, effect of expansins on the lost ofmechanical strength of paper was measured by stess relaxation assay. Forstress relaxation measurements, the paper was incubated in variouspretreatment solutions and assayed while still wet, in the standardmethod.

In extensometer assays S1 protein induced a brief period of extensionprior to rapid breakage of the paper, an effect not seen in incubationswith buffer alone or with boiled protein. Incubations of paper stripswith cellulase showed a similar effect. However, it required at least100 mg of cellulase to weaken the paper sufficiently to be detected inthis assay, whereas only 5 mg of S1 protein produced similar effects. Inparallel experiments, cellulase (100 mg/mL) was shown to release 25 mmolreducing equivalents per mL during a five h incubation with Whatman No.3paper. In contrast, no sugar release was detectable in similar assaysusing S1 and S2 protein (both 5 mg/mL). The results are presented inFIG. 6.

S1 protein (5 mg/mL) caused a marked increase in the relaxation rate ofpaper strips, while cellulase (100 mg/mL) had almost no affect. In bothextensometer assays and stress relaxation tests, S1 protein which hadbeen boiled for two minutes did not induce the effects apparent withnative S1 proteins, indicating that the effects were associated with theactivity of the protein. Similar results were obtained in assays with S2proteins (data not shown).

These data indicate that expansins substantially weaken cellulose paperin a manner which does not involve the hydrolysis of cellulose. Sincethe structural integrity of cellulose paper appears to result fromhydrogen bonding between cellulose fibrils, it seems likely that theseproteins weaken paper by disrupting these associations.

EXAMPLE 11

Effect of Cucumber Expansins on Slick Paper

In order to test whether a protein preparation containing cucumberexpansins can disrupt slick paper, in a similar fashion as it acts onpure cellulose paper the following experiments were conducted. Slickpaper was obtained from three sources: (a) the pages of "Nature"magazine (unprinted margins were used), (b) a commercial catalog forchromatography equipment (c) a colored advertising insert (Hills) in theSunday paper. Paper strips of 2-3 mm width were cut and hung in ourextensometer, such that a 5-mm long strip was put under 20-g force. Thebuffer contained 50 mM sodium acetate, pH 4.5 with or without added C3protein (about 20 ug per paper strip was used). The length of the paperstrips was recorded for up to 5 h. Four samples of each paper were usedwith each treatment (+/- protein). The untreated (control) paper showedvery little change in length, and maintained its mechanical strengththroughout the test period. In contrast, the treated papers began toextend and quickly broke once extension began. The time for breakageafter addition of protein varied from 45 min to 2.5 h, but all samplesbroke. FIG. 7 illustrates the typical extension.

EXAMPLE 12

Effect of Snail Expansin -like Proteins on Cellulose Paper

A stress relaxation assay was performed as described in Example 10.Increased relaxation in time domaines of 10-100 msec (faster relaxationin comparison with controls) proved the ability of snail expansin-likeproteins to weaken the mechanical strength of paper.

EXAMPLE 13

Preparation of Antibodies to Cucumber Expansin cEx-29

Antiserum with specific recognition of this protein was raised in afemale New Zealand White rabbit (Li and Cosgrove, in preparation) bysubcutaneous injections of cucumber Ex29 with Freund's adjuvants (Harlowand Lane 1988). Serum dilutions in the range of 2000:1 to 4000:1 provedoptimal for Western analyses of cucumber and oat proteins.

EXAMPLE 14

Western Blot Analysis of Immunoreactivity between Cucumber and OatExpansins

We performed western analysis of oat coleoptile proteins probed withantiserum against cucumber Ex29. Results are presented in FIG. 17.Comparison of lane 1 (crude wall protein) with lane 2 (crudeprotoplasmic protein) shows that the antiserum recognizes an Ex29-likeprotein (arrow) specifically bound to the coleoptile cell wall.Comparison of lane 3 (crude wall protein) with lane 4 (purified proteinwith extension activity) shows that the Ex29-like protein co-purifieswith the extension activity. Lane 1 was loaded with 15 μg of crude wallprotein (ammonium sulfae precipitate of 1 M NaCI extract). Lane 2 had 15μg of the soluble protoplasmic protein fraction. These samples wereseparated on the same 4-20% gradient SDS polyacrylamide gel, blottedonto nitrocellulose, and probed with rabbit antiserum against cucumberEx29. Lane 3 was loaded with 0.2 μg of crude wall protein and Lane 4 had0.2 μg of active oat wall-extension protein purified sequentially byDEAE Sephadex and CM-HPLC. These samples (3-4) were separated on thesame 14% SDS polyacrylamide gel, blotted onto nitrocellulose, and probedwith antiserum. This assay, with minor variations, was carried out fourtimes with similar results.

EXAMPLE 15

Western Blott Analysis of Immunoreactivity between Expansin-like Proteinfrom Snail and Cucumber Expansin cEx-29.

FIG. 20 presents a Western blot of active HPLC- separated proteinfractions from snail acetone powder, probed with antibody PA1 which wasraised against cucumber expansin- 29. The active fractions show astriking band at about 26 kD, which is similar to (though slightlysmaller than ) cucumber wxpansin-29. This results provide strongevidence that the wall extension activity found in the snail acetonepowder is due to a protein with similar antigenic determinants ascucumber expansin-29.

FIG. 21 presents a Western blot of the proteins from Helix aspersafeces. probed with the antibody PA-1. The single band indicates thepresence of an protein antigenically similar to cucumber expansin-29.

EXAMPLE 16

Effects of Urea on Plant Cell Wall Extension

This example shows the effects of 2 M urea on native cell walls, wallsreconstituted with expansin, and heat inactivated walls. Native cellwalls or heat-inactivated cell walls from cucumber hypocotyles wereobtained as earlier described and clamped in the extensometer in 50 mMsodium acetate at pH 4.5. At about 20 minutes, an S 1 fraction ofcucumber expansin (10 μg/mL) was added to one set of heat-inactivatedwalls. At about 45 minutes the incubation solutions were replaced with 2M urea buffered with 50 mM sodium acetate, pH 4.5. The aforesaidsolutions also contained 2 mM dithiothreitol to stabilize the expansinactivity. Results of this experiment are presented in FIG. 22. Data arerepresentative of four (4) trials for each respective treatment. Thedotted line of FIG. 22 represents native cucumber cell walls.

These data show that 2 mM urea acts synergistically with expansins toenhance the extension rate of native cucumber cell walls andheat-inactivated cell walls reconstituted with purified expansins. Asindicated in the trace labeled "heat-inactivated" in FIG. 22, urea alone(without active expansins in the cell walls) has little effect.

These results support the contention expansins disrupt hydrogen bondingbetween cell wall polymers.

EXAMPLE 17

Identification of Expansins in Various Plant Materials

Using the methods earlier described herein for extraction of expansinsfrom cucumber and oat materials, quantities of the following plantmaterials were treated:

(i) broccoli flower stalks (Brassica oleracea italica);

(ii) celery peticles (Apium graveolens);

(iii) tomato leaves (Lycopersicum esculentum);

(iv) cotton fibers (Gossypium); and

(v) corn coleoptiles (Zea mays).

Expansins were identified and confirmed by use of the antibody assaydescribed herein (having common antigenic epitomes with the antibodyagainst ex29), and the extension activity analysis described herein.

EXAMPLE 18

Use of expansins to enhance the accessibility of Cellulose to Cellulaseand related Enzymes and Processes.

Expansins are shown to make cellulose more accessible to the degradativeaction of cellulases, thereby synergizing or enhancing the activity ofcellulases. Cellulases encompasses any enzyme which hydrolyzes celluloseinto smaller units. It is likely that this newly discovered effect ofexpansins may also enhance the accessibility of cellulose for chemical,biochemical, or biophysical modification. The data shown in FIG. 23provide supporting evidence for the present invention that expansins inthe presence of cellulase act synergistically to hydrolyze celluloseinto smaller units. This data suggests that classes of expansins,classes of enzymes possessing expansin-like activity, and biologicalorganisms genetically modified with an expansin gene or a generesponsible for expansin-like activity will be synergistic withcellulases to modify cellulose.

Cellulose is the most abundant polymer on earth. It has numerous uses inmany industries, including: as a fiber in the paper/pulp and textileindustries, as a starting material for manufacture of films, coatings,membranes, thickeners, and as a biomass source for alcohol production.

The problem with cellulose is that the substance is a microcrystallinefiber that is mechanically and chemically very tough and resistschemical and biological degradation. Chemical means of solubilizing ordegrading cellulose are available, but they have disadvantages: harshconditions or chemicals are required or they modify the constituentpolymers in undesirable ways. Although numerous cellulases are in usecommercially to breakdown cellulose, they suffer from relatively pooreffectiveness because the enzymes cannot gain ready access to thecrystalline polymer.

If a means could be found to break open, decrystallize, or by anotherenzymatic mediated process to modify cellulose under milder conditions,this might have considerable practical benefit to industries thatprocess cellulose or manufacture products utilizing cellulose.

To test whether expansins can modify cellulose or "open up" thecellulose crystal and make it more available to degradation bycellulase, a microcrystalline form of cellulose (Avicel) was incubatedin the following solutions:

1. pure buffer,

2. buffer+expansin,

3. cellulase, or

4. expansin+cellulase

50 mg of Avicell (microcrystalline cellulose) was placed in 1 mL buffer(mM sodium acetate, pH 4.5) with expansin alone ("C3" fraction, about 50μg per mL), or cellulase alone (100 μg of Trichoderma cellulase fromBoehringer, purified to remove sugars), or expansin plus cellulase (50μg C3 expansin and 100 μg Trichoderma cellulase). After 1 h and 2 h,aliquots were removed and assayed for sugars with standard reducingsugar assay. This experiment was carried out twice with nearly identicalresults.

The release of sugars from the cellulose was assayed as a measure ofcellulose degradation. The results as shown in FIG. 23 herein attachedindicate that expansins by themselves do not release sugars, but theyenhance the effectiveness of cellulase by 50%. These results provide aninitial example of expansins synergistic effect with cellulase oncellulose. Other methods of treatment or use of specific expansins,enzymes possessing expansin-like activity, or genetically modifiedorganisms possessing expansin gene(s) or gene(s) that have expansin-likeactivity may modify cellulose by a number of various actions, includingbut not limited to chemical, biochemical, or biophysical activity. Suchvariations are anticipated to have numerous commercial utility, whichmay include but not be limited to the following, the modification oralteration of cellulose in the production of commercial products orintermediates utilized in production of paper/pulp, textile, fodder,biomass alcohol, films, coatings, thickeners, agricultural and/or foodapplications.

We know from previous work that expansins bind to cellulose and thatthey can disrupt hydrogen bonding between cellulose microfibrils(McQueen-Mason and Cosgrove, Plant Physiology 107: 8-100; Proc. Nat.Acad. Sci. 91: 6574-6578). These new results show that expansins arealso able to enhance cellulase degradation of the microfibril. Thestrong conclusion is that expansins can open up the microfibril toenzymatic attack by splitting off the individual polymers that make upthe crystal and making them available to the cellulase. Because theglucan polymers that make up cellulose are held in the crystal by bothhydrogen bonding and hydrophobic bonding, the results mean thatexpansins can disrupt both types of bonding. As additional research isperformed, other methods of action may be discovered, which havepotential commercial utility. However, it is understood that thedisclosed embodiment is merely illustrative of the invention, which maybe embodied in various forms. Accordingly, specific details disclosedherein are not to be interpreted as limiting, but merely as a supportfor the invention and as appropriate representation for teaching oneskilled in the art to variously employ the present invention in anyappropriate embodiment.

This invention suggests that the expansin proteins might find largescale use in processes for degrading or chemically modifying celluloseitself or altering the physico-chemical characteristics of cellulosicmaterials. Moreover, by genetically engineering plants to express largequantities of expansins in their mature walls, it is likely that thatcellulose in these walls will have altered physico-chemical propertiesand will be more accessible to chemical and enzymatic modification andattack. This might make such genetically engineered plants of greateruse for paper/pulp, fodder, biomass alcohol production, or textiles andthe other commercial applications cited above.

Alteration of the specific expansin, cellulase, and source and type ofcellulose are within the scope of this invention. Such variation mayhave significant, commercial utility. These results may vary because of,but not limited to, the following factors: substrate specifications,solubilities, pH optima, and other biochemical and/or biophysicalproperties, but expansins in concert with cellulase will nonethelessenhance or synergistically impact cellulose degradation.

What we present here is a method using expansin(s), enzymes possessingexpansin-like activity or genetically modified organisms possessingexpansin gene(s) or genes that have expansin-like activity in thepresence of a cellulase to modify cellulose by a number of variousactions, including but not limited to chemical, biochemical, orbiophysical activity.

It is noted that previous research has subjected expansins to a pHranges from 3.0 to 8.0 and in the presence of a number of differentbuffers, such as sodium acetate, HEPES, EDTA, sodium bisulfite,MES-NaOH, PBST and Tris-HCL. Variations of reactant concentrations,buffer, and pH are possible and could be optimized for a given set ofreaction conditions to facilitate the reaction. Obviously, extreme pHs,such as 1 or 14, would be inappropriate, since they would eitherdenature the cellulase or expansin or inhibit the enzymatic reaction.The type of buffer to be used will depend upon the desired pH and otherphysiological considerations. The above listed buffers should beconsidered an example and not specifically limiting to this group.

FIG. 23 shows that the cellulose degradation curve is measured by therelease of sugars from the cellulose. It shows that the cellulosedegradation curve of cellulase and expansin is very different from thatof cellulase only, or of expansin only, and they do not have asuperimposed relationship. This shows that cellulase and expansin have asynergistic effect in degrading or modifying cellulose. The recommendedfinal concentration and type of cellulase and of expansin will depend onthe specific requirements of the reaction, e.g. in some cases 100 ug oftrichoderma cellulase and 50 ug of cucumber C3 expansin may be adequatebut much higher and lower concentrations and different ratios and typesof cellulase and of expansin may be prescribed and are within theteaching of this invention.

The length of reaction time to effect the desired alteration ofsubstrate may be altered and such time variation is anticipated.Conversely, variations of the type and concentration of expansin and ofcellulase along with the specific physiological conditions will affectthe rate of reaction.

In summary, cucumber expansins appear to associate with the cellulosefraction of the cell wall. They do not exhibit polysaccharide hydrolysisunder a number of assay conditions and they do not cause a progressiveweakening of the wall. Expansins also appear to disrupt hydrogen bondsas particularly noted with cellulose paper.

Isolation of new enzymes often opens new possibilities of application ofsame. Although the potential application in paper industry have beenemphasized in this disclosure, numerous other directions of use can beimagined. For example expansins could be used for processing ofpolysaccharides for control of physical properties. Hydrogen bonding isan important determinant of many physical properties of commercialproducts containing polysaccharides. Expansins may be incorporated intothe polysaccharide products to modify hydrogen bonding and therebymodify the physical characteristics of the products. Examples includecontrol of the viscosity and texture of polysaccharide thickeners usedin foods and chemical products, control of stiffness and texture ofpaper products; and control of mechanical strength (e.g. tear strength)of paper products.

The present invention is believed broadly applicable to alteration ofvarious physical properties of polysaccarides. While plantpolysaccarides represent a preferred embodiment of the invention,expansins of the invention are believed useful catalytic proteins forthe treatment polysaccarides from a variety of sources (i.e., synthetic,bacterial or other microbial system, etc.). While it is reasonable tomake a strong claim that expansins primarily operate by disruption ofhydrogen bonds, the invention is not necessarily limited to this mode ofaction. Results concerning altered physical properties of polysaccardiesare nonetheless produced by use of the novel expansins of the invention.

Expansins may be used for de-inking paper, which is a significantlimitation in current paper recycling operations. In this application,expansins may help loosen the bonding between surface polymers, whichare stuck to the ink, and the remainder of the cellulose fibers. Also,in the paper industry expansins may prove useful for large scale paperdissolution, or perhaps for alteration of the mechanical properties ofdry paper. Treatment of dry paper could produce paper with novelproperties.

Expansins may be used in combination with other chemicals or enzymes toimprove various processes. For example, a major limitation in ethanolproduction from biomass is the degradation of cellulose. Expansins inconcert with cellulase may act synergistically in the breakdown ofcellulose. If expansins help celluloses gain access to glucan chainsthat make up microfibrils, then they could speed cellulose action. Also,delignification is a major problem in industrial uses of many plantfibers. To solve this concern, expansins may be used with lignases(peroxidases) for synergistically de-lignifying plant fibers. Expansinsmay also be useful in various bioremediation applications, either aboveor in combination with other biological or chemical materials.

Expansins have been found in cotton fibers and are probably found innative cell walls, but are likely not to be present in processed cottonfiber walls. Accordingly, expansins may be useful to the textileindustry by virtue of alteration of either the wet or dry mechanicalproperties of cotton flax, or other natural fibers.

It is known that certain fungal cell walls (i.e., Phycomyces) bearchemical resemblance to the structure of plant cell walls. Also, certaininsects have chitin structures or walls (i.e., Manduca cuticle) thatresemble fungal walls in some of their biochemistry. Expansins maymodify chitin properties. Accordingly, expansins may be useful forinsect control or as anti-fungal agents, either alone or in combinationwith other insecticidal or fungicidal agents.

A further use of expansins could be for altering the mechanical strengthof gels or otherwise affecting the gelling or other properties of gels(i.e., gelatin, gellum-gum/phytagel, agar, aarose, etc.). Such materialare useful agents in foods, cosmetics, and other similar materials.Since hydrogen bonding is important for such gels and in view of thebelief that expansins alter hydrogen bonding of wall glycons, expansinsmay alter the gelling properties of various gels.

An additional use of expansins may involve alteration of aggregation ofhemicelluloseA and solubilized cellulose. In the event that expansinsappropriately affect such aggregation, they may prove useful forindustrial processes involving these materials, including celluloseprocessing and film making.

Isolation of a novel protein allows one to attempt the cloning of thegene coding for this protein using the standard approach of establishingamino acids sequence of a fragment of the protein and designingoligonucleotides to screen the cDNA library. When cloned, the gene forone or more expansins will need to be expressed in a bacterial or othersystem to obtain sufficient quantities for the commercial usefulness ofthe ideas listed above. Cloning will also be a necessary first step forthe commercial uses requiring genetic manipulation of the protein intransgenic plants.

Engineering of plant cell growth for agricultural uses can becomereality. We have evidence that expansins are important for endogenousgrowth of plant cells. One can envision several commercial applicationsof expansins. One such application includes increase of the growth ofspecific organs and tissues by selective enhanced expression ofexpansins during plant development. Control of selective expressionwould be by any means such as application of specific chemicals thatpromote transcription of expansins or by insertion into the genome ofplants specific artificial genetic constructs (i.e. appropriatepromoters, enhancers, structural genes and associated elements) toeffect organ-specific, tissue-specific, and/or chemical-specificenhancement of expression of the introduced or endogenous genes for theexpansin proteins. Examples of use might include enhancement of fruitproduction (grapes, citrus fruits, etc.), enhancement of leaf growth(lettuce, spinach, cabbage), enhancement of stem or petiole growth(sugarcane, celery, flower stalks), and enhancement of root growth.Tissues engineered to grow by expression of expansins might haveenhanced desirable traits (size, succulence, texture, durability).

Another direction of controlled growth would include decrease of thegrowth of specific organs and tissue by reducing the expression oreffectiveness of endogenous expansins. The control of expression wouldbe by any means such as application of specific chemicals that reducetranscription of expansins or by insertion of antisense geneticconstructs that reduce mRNA levels of endogenous expansin genes, or bymanipulation of the genetic control elements that regulate expression ofendogenous expansin genes. Examples of such use might include dwarfingof stems for enhanced mechanical stability and genetic pruning,stunting, or elimination of undesirable plant organs.

Similar approach could be utilized to control the cell size in plantcell tissue or cell cultures used in bioreactors or for production ofuseful chemical agents. Control might be effected by addition ofexogenous expansins or inhibitors of expansin action, by geneticregulation of endogenous expansin genes and their products, or byregulation of artificially inserted genes for expansin proteins, forantisense constructs against endogenous expansin mRNAs, or for proteinsthat regulate or modify expansin action.

Thus, while we have illustrated and described the preferred embodimentof our invention, it is to be understood that this invention is capableof variation and modification, and we, therefore, do not wish or intendto be limited to the precise terms set forth, but desire and intend toavail ourselves of such changes and alterations which may be made foradapting the invention of the present invention to various usages andconditions. Accordingly, such changes and alterations are properlyintended to be within the full range of equivalents and, therefore,within the purview of the following claims. The terms and expressionswhich have been employed in the foregoing specification are used thereinas terms of description and not of limitation, and thus there is nointention in the use of such terms and expressions of excludingequivalents of features shown and described or portions thereof, itbeing recognized that the scope of the invention is defined and limitedonly by the claims which follow.

Thus is described our invention and the manner and process of making andusing it in such full, clear, concise, and exact terms so as to enableany person skilled in the art to which it pertains, or with which it ismost nearly connected, to make and use the same.

What is claimed is:
 1. A composition comprising an isolated, purifiedand salt-soluble polypeptide in an acid medium, wherein the polypeptidehas an amino acid sequence with a molecular weight of about 29-30 kD asmeasured by SDS-PAGE, and induces expansion of inert plant cell wallmaterial.
 2. A composition according to claim 1, wherein the pH of theacid medium is in the range of about 3.5 to 5.5.
 3. A compositionaccording to claim 1, additionally comprising a sulfhydryl reducingagent.
 4. A composition according to claim 1, wherein the expansion isirreversible.
 5. A composition according to claim 1, wherein thepolypeptide is of plant origin.
 6. A composition according to claim 5,wherein the polypeptide is derived from a plant family selected from thegroup consisting of cucumber, oat, broccoli, celery, tomato, cotton,flax, cabbage and corn.
 7. A composition according to claim 1, whereinthe polypeptide is of snail origin.
 8. A composition according to claim1, wherein the polypeptide is derived from the cell wall material of thegrowing region of a plant.
 9. A composition according to claim 8,wherein the plant is selected from the group consisting of cucumber,oat, broccoli, celery, tomato, cotton, flax, cabbage and corn families.10. An isolated and purified polypeptide having an amino acid sequencewith a molecular weight of about 29-30 kD as measured by SDS-PAGE, andwhich induces the extension of plant cell wall material in the presenceof an acid.
 11. A polypeptide according to claim 10, that is isolatedand purified by extraction from plant cell wall material.
 12. Apolypeptide according to claim 11, wherein the extraction is from agrowing region of the plant cell wall, and wherein the acid has a pH inthe range of about 3.5 to 5.5.
 13. A polypeptide having the ability toinduce the extension of plant cell wall material, comprising an aminoacid sequence that is recognized by an antibody raised against a proteinhaving a molecular weight of about 29-30 kD as measured by SDS-PAGE, andderived from the growing region of cell wall material isolated from aplant selected from the group consisting of cucumber, oat, broccoli,celery, tomato, cotton, flax, cabbage and corn.
 14. A polypeptideaccording to claim 13, wherein the protein is derived from cucumberhypocotyls.
 15. A polypeptide according to claim 14, wherein the proteinis cEx-29.
 16. A polypeptide according to claim 13, wherein theextension of plant cell wall material is irreversible and occurs in thepresence of an acid.
 17. A polypeptide enzyme according to claim 13having the ability to weaken the mechanical strength of paper.
 18. Apolypeptide according to claim 17, wherein the paper contains cellulose.19. A composition according to claim1 having the ability to weaken themechanical strength of paper, comprising a polypeptide in aqueoussolution with an acid, wherein the polypeptide includes an amino acidsequence that is recognized by an antibody raised against a proteinwhich induces the extension of plant cell walls, has a molecular weightof about 29-30 kD as measured by SDS-PAGE, and is derived from thegrowing region of cell wall material isolated from a plant selected fromthe group consisting of cucumber, oat, broccoli, celery, tomato, cotton,flax, cabbage and corn.
 20. The composition according to claim 19,wherein the pH of the acid medium is in the range of about 3.5 to 5.5.21. The composition of claim 20, wherein the acid is at least one ofsodium acetate and urea.
 22. The composition according to claim 20,additionally comprising a sulfhydryl reducing agent.
 23. An isolated andpurified polypeptide having a molecular weight of about 29-30 kD asmeasured by SDS-PAGE, and having at least one of the followingproperties in the presence of an acid: (i) restoring extension activityin heat-inactivated cell walls; (ii) disrupting hydrogen bonds betweencellulose fibers and (iii) weakening the mechanical strength of paper.24. A composition comprising an isolated and purified polypeptide ofclaim 23 in an acid medium.
 25. An isolated and purified polypeptidehaving a molecular weight of about 29-30 kD as measured by SDS-PAGE andhaving at least one of the following properties in the presence of anacid: (i) restoring extension activity in heat-inactivated cell walls;(ii) disrupting hydrogen bonds between cellulose fibers and (iii)weakening the mechanical strength of paper, wherein said polypeptide isof oat, broccoli, celery, tomato, cotton, flax, cabbage or corn origin.26. A composition comprising an isolated and purified polypeptide ofclaim 25 in an acid medium.